11 B.B.2 AWAROS B Official 5 B.B.2.2 Awards IZS Umbria e Marche Organic contaminants unii in the Centro di Ancona Head of the contaminants Organic Unii , Referenza per il controllo chimico e microbiologico dei I. molluschi bivalvi.- ANO HONORS H Index and Honors 1997 Degree in Chemistry al the Universily of Bologna 1997 Master af Philosophy (Science) al the University cf Birmingham 1997 Qualification as chemisl for the entry to the chemisls list, University cf Bologna 1997 Assignment of a PhD position al the University of Venezia associated with the University of Ferrara Specialization in Chemistry and Food Technologies allhe University of Perugia.:"'0.; \ Other CV informatioo Research in organic synthesis applied lo supramolecular chemistry under the supervision of Prof J.F. Sioddart at the School of Chemistry of the University of Birmingham Research in photochemistry and electrochemistry in the group of Prof Vincenzo Balzani at the University of Bologna The PhD grant was funded by the European Vinyl Corporation (EVC) Porto Marghera, Venezia and the activity was in controtling the levels of Persistent Organic Poltutants (PCDD/F, PCS, IPA) in industriai emissions. Since the end of 1997 the activities focused on analyticai chemistry, applied to environment and food, and dealing, particularly, with organic contaminants. Experienced in analytical melhods development and validation using PLE, SPE, SPME, GPC sample preparation techniques and GC with selective detectors, GC-MS, HPLC-DAD for the instrumental analysis. On-going education was achieved atlending numerous training courses in the field of analytical chemistry and fooosafety and national and interna'tional conferences. Author or co-author of several scientific publications (intemational or national journals and conferences proceedings), mostly 00 analytical chemistry applied to food safety. Lecturer in several training courses of the nationai health system and of the universily dealing manly with persistent organic contaminants, pesticides, food additives and biogenic amines. Partner in National granted research projects (Ricerche Correnti) and Principai Investigator in 2001 (Ricerca Corrente). B BIOGRAPHICAL B.A Personal SKETCH CONTRIBUTORS NAME INSTITUTION ANO POSITION TITlE Monia Perugini Università degli Studi di Teramo, Facoltà Medicina Veterinaria. Confirmed researcher statement In vivo pade toxicological studies using zebrafish as mode!. B. B Positions and Honors B.B.1 POSITIONS B.B.2 AWAROS B.B.2.1 Offidal 6 INSTITUTION OIVISION LOCATION POSITION FROM TOYEAR YEAR Università degli Studi Veterinary Teramo PhO di Teramo Pha~~~~gy and Toxicol Unit Università degli Studi Veterinary Teramo researcher di Teramo Pharmacology and Toxicoloav Unit ANO HONORS H Index B.B.2.2 Awards and Honors 2000 Degree cum laude in Veterinary Medicine at the University of Teramo 2004 PhO in Veterinary Pharmacology and Toxicology at the University or Napoli 2005 Researcher in Veterinary Pharmacology and Toxicology, University of Teramo 2008 Confinned researcher in Veterinary Pharmacology and Toxicology, University of Teramo Other CV information Research in fooomicrobiology in the group of Prof A. lanieri, at the University of Teramo PhO stage in ecolossicology and bioassays lechniques, under the supervision of Dr. E. His, laboratory of "Environnement Ressources" IFREMER, Arcachon (France) Teaching in: Residual Toxicology of AnimaI Food Products - Risk Assessment; Veterinary Pharmacoiogy: Velerinary Pharmacology and Pharmaceutics at the University of Teramo Reviewer for international joumals: Environment Internalional, Environmental Poltution, Meat Science, Estuarine, Coastal and Shelf

12 Science, Fresenius Environmental BulJetin, Joumal of Agricultural, Food Chemistry, Chemosphere, Biological Traee EJement Researeh, Environmental Seience and Pol1ution Research, Food and Chemical Toxicology, Journal of Food Nutrition and Research, Polish Journal of Environmental Studies. Since the end of 2000 the research activity was focused on toxicology, ecotossicology and analytical chemistry particularly organic contaminants in fooo and environment. Author or co-author of 57 scientific publications of whìch 1B in inlernational scientific journals and 30 in proceedings of inlernational or national congresses. Author of 7 chapters of international books and of 1 chapter of an Italia n veterinary medicine book. Experienced in analytical ehemistry: method development and validation using PLE and SPE sample preparation teehniques and GC-MS and HPLC for the instrumental analysis. Experienced in ecotossicology: environmental bioassays techniques and appticalion. Toxieity studies of persistent organic compounds using oyster and sea urchin larvae. Experienced in pharmacology: in vitro evaluatlon of compounds altering the funetion of smooth musele in the bronchial tissue of horses with chronic obstructive pulmonary disease. In vitro evaluation of the contractile response of intestinal strips of rainbow lrout to serotonin and histamine and the effect of eosinophil degranulation. On-going education was achieved participating to numerous workshops and seminars in the field of food toxicology and fooosafety and to national and international conferences. Partner in Programmi di ricerca di Rilevante Interesse Nazionale (PRJN) in B BIOGRAPHICAL SKETCH CONTRIBUTORS NAME Sabrina Tait 8.A Personal statement In vitro PBDEs toxicological studies on human celi tines 8.B Positions and Honors B.B.l POSITIONS INSTITUTION ANO POSITION TITLE Istituto Su eriore di Sanità - Conctract researcher B.B.2 AWAROS 8.8,2.1 Official H Index 3 INSTITUTION OIVISION LOCATION POSITION FROM TOYEAR YEAR Istituto Superiore di Sanità Enterovirus Unit Roma Contract Researcher Istituto Superiore di Food and Toxicology Roma Contract Researcher ~ Sanità Unit ANO HONORS Awards and Honors 2000 Degree in Chemistry at La Sapienza University of Rome 2000 Qualifìcation as chemist for the entry to the chemists lisi, La Sapienza University of Rome 2005 Second level Master cum laude in Bioinformatics al La Sapienza University of Rome Other CV information Research in natural and synthetic antivirals modes of aclion against enteroviruses under the supervision of Doci- Lucia Fiore al lhe Enterovirus Unit of the Istituto Superiore di Sanità Research in endocrine disruptors effects and modes of action in the Food and Toxicology Unit (headed by Prof. Alberto Mantovani) of the Istituto Superiore di Sanità Since the end of 2005 the research activity was mainly focused 00 the study of endocrine effects 00 reproduction and development, especiajly on target organs like brain, placenta and male extemal genitalia following exposure, respectivety, lo the pesticide chlorpyrifos, the plastic addictive Bisphenol A and differenl mixtures of Polychlorinated Biphenyls. Since 2008 involved in the PREVIENI Project founded by the Ministry of Environment, whose aim is to perform a biomonitoring study in 4 different areas by analysing some endocrine disrupting levejs in serum of couples affected by infertility, their clinical profite and some biomarkers of earty exposure in leucocytes at gene expression level. Experienced in celi and molecular biology techniques such as Gel electrophoresis, Western Blot, ELISA, Immunocytochemistry, PCR and Real-time PCR, DNA Sequencing, RNA transfection, recombinant DNA, Microarray. On-going education was achieved participating to numerous workshops and seminars in the field of food toxicology and food safety and to national and intemational conferences. Author or co-aulhor of several publications (international or nalional joumals and conferences proceedings) on antivirus research ( ), reproductive/developmental toxicology and fooosafety ( ). Involved in several Ministry of Health (Ricerche Finalizzate), an ISS-NIH, and several European granted research projects within the 7th Framework programme. C Rationale,AIMS C1 BackGround ANO IMPACT Polybrominated diphenyl ethers (P8DEs) are a class of emerging substances used worldwide as f1ame relardants in electronic appliances, textiles, furnishings and various other consumer products. There are theoretically 209 PBDE congeners, although the commerciai products predominantly consist of: penla-bdes (PeBDE), used in epoxy or phenol resins, polyeslers, polyurethane foam and textiles; octa-bdes (OcBDE), employed in

13 /<>. '~;~;!G;':~'... :)~..,\ /,.~<,". acrylonitrile butadiene styrene, polycarbonate and thennoset; deca-bdes (DeBDE) used in most!ypes of synthetics material~_'i'~~luding\exti!'esand ':',:~1 polyester far printed circuit boards [1J. \ ;;"" Since PBDEs are added lo the products rather than be chemicaljy bound lo them, they can be slowly and continuously releaskdduring their \'. ' manufacturing, while in use, or after their disposal. The disposal of plastics is probably the mai n source of PBOEs in the environment and, as a '-'r. resull of their ehemical stability, they are globally Iransported and distributed. PBDEs are persistent and have very low water solubìlity,;h.igh,binding. affinity lo particles and tendency to accumulate in sediments. The presence of PBDEs in biota is likely to be a result of their lipophilìciìya~d'"''''::'~'~' resistance to degradation {1, 2]. Furthermore, it has been demonstrated that congeners with less than seven bromine atoms are more bioaccumulative than lhe heavier PBDEs, exhibiting large biomagnification faclors through the food chain. Generally, PBDEs seem to cause toxic effects al low doses. The most sensitive end poi n! of PBDEs toxicity in vivo is related to thyroid function, wilh induction of thyroid hyperplasia and alteration of thyroid hormones production in rats and mice [3, 4). Moreover, PBDEs share with co-planar polychlorinated biphenyls (PCBs) and dioxins the Aryl Hydrocarbon Receptor (AhR)-mediated induction of some hepatic cytochrome P450 enzymes, such as 1A1, 1A2 and 3A1 [5,6]. Several pure di. to hepta-brominated PBDEs congeners are able to interact with AhR in vitro as agonists or anlagonists in a congener-specific manner. Relalively recenl reports indicate that exposure lo low concentrations of these chemicals may result in irreparable damage lo the nervous and reproductive systems. Earty-life exposures lo PBDEs at relatively low levels can also lead lo delays in sensory-motor devetopmenl, hearing deficits, as well as changes in activity levels and fear responses f7j. An ideai organism for the study of vertebra le developmental biology is zebrafish, (Danio rerio) which has favorable characleristics and il is considered a gooo in vivo frsh model to 'Invesligale endocrine disruption and neurobehavioral effecls exerted by environmental contaminants. Sioce liver is a main targel organ of PBDEs action, ao elective modello study in vitro toxicity of these compounds, especially at molecular level. is represented by human liver celliines. Moreover, due to the primary importance of the digestive system as food and food contaminants processar, the use of human colon celllines may help to elucidate further PBDEs mechanism not yet investigated. So far, no Toxicily Equivalency Faetors (TEFs) have been defined for PBDEs congeners.1t is, therefore, needed to explore comprehensive toxicological parameters to establish TEF values in order to assess P8DEs risks for human heallh. Among food, fish and shellfish, highly contribule lo P8DEs human intake, especially if characterised by high fat content and grown in contaminated areas [8]. Mussels are a significant vehicle of dietary exposure because lhey can concentrale chemicals ( times) as a result oftheir ability lo filler large volumes of water. Several studies were performed in emerging counlries Iike China, India and Korea,. where a massive disposal of electronic equipment is carned over, as wetl as in northern Europe and USo in order lo analyze PBDEs levels in mussels and sediments. So far, no dala are stili available conceming PBDEs contamination of mussels grown on Italia n coasts and specffically in a landlocked sea lìke the Adriatic. To detect PBDEs in biologicat samples it is needed lo reach sub-ppb levels, therefore sensilive and specific analytical procedures are required. Gas chr~matography coupled to mass spectrometry is the elective analytical lechnique in PBDEs analysis. Recently, in the field of micro-pollutants analysis, the use of MS/MS has been considered a valuable approach altowing lo minimize malrix interferences and reach very Icw detection limils with good precision enabling sufficienl sensitivity and selectivily [9,10]. [1] De Wit, C Chemosphere, 46: [2J Alaee, M Environment Intemalionat, 29: [3] Fowles, J.R Toxicology, 86: [4] Damerud, P.O Organohalcgen Compounds, 29: [5] Meerts, I.A.T.M Environmenlal Health Perspectives, 109: [6] Fery, Y et al Toxicotogy, 264: 45-51_ [7] Branchi, I Neuro Toxicity, 23: [8] Domingo, J.L Journal of Chromatography A, 105: [9) Covaci, A Environment Inlemalional, 29: [.10JVonderheide, A.P Microchemical Journal, 92: C2 Aims The overall objective of lhis inlerdisciplinary project is to improve Ihe known-how and the risk assessment on brominated f1ame retardants as PBDEs, for human and wildlife health. The developmenl of new lools for detection and monitoring of PBDEs in mussels (Mytilus galloprovincialis) as well as the evalualion of their bioaccumulation, reproductive, neurological, hepatic and enteric effecls on in vivo and in vitro models, will be the principal aim of the project. For this purpose, the project will use ao integrated approach by combining chemical, cellular, molecular and histological lechniques for which each cf the contributors has recognized experience. By integrating information on exposure. fate and toxicity af P8DEs, this project aims to provide a highly significant contribution lo integrated risk assessment of this class of compounds, for humans and aquatic environment safety. Moreover, the integralion of data on a multidisciplinary basis will serve to propose C3 Main Expected Resulls and Impact a TEF approach for PBDEs toxicity c1assifrcation The overal! objective of the project is to provide comprehensive information on PBDEs spread in the Italian aquatic environment Ihrough the investigalion of mussels conlaminatian as well as to fili some gaps on the knowledge of their toxicity_ Specific expected results of this study are to: - implement validated and high throughput analytical methods for the delermination of PBDEs at sub-ppb level in mussels, fish and feed; - define the background concentrations and congeners profile of PBDEs in mussels sampled along the Italian coast and particularly in the Adriatic Sea; - enable comparison with PBDEs international data; - investigate the PBDEs concentrations at specific potential point source like sewage Ireatment plant outfalls, rivers outlel, induslrialised areas etc; -,evaluate possible developmenlal malformations, changes in behavlaur and de.ath incidence foltowing PBDEs exposure during the early-life stages of zebrafish, when fish are usually mosl sensitive to organic contaminants. - compare the toxicity of a lower brominated congener (BDE-47) and a higher brominated congener (BDE-209), moreover investigating possibte

14 addictive/synergistic effects exerted by the PBDEs mixture of the 8 EFSA dielary relevanl congeners; - investigate the effect of freeze-dried mussels intake as a sum of toxiclprotective actions; - study of the zebrafish liver, gonad and lhyroid histology following exposure lo PBDEs; - identify possible innovative cellular biomarker of exposure and/or effect as rodlet cells and macrophage aggregales (MAs and RCs) in zebrafish; - identify further and stili not documented liver, as weu as colon, human molecular targets and modes of action following exposure to PBDEs of human hepatic (HepG2) and enteric (DLD-1) celliines Ihrough the exploration of their complete gene expression profile; - indenlify early biomarkers cf exposure on human liver and colon celllines following both PBDEs and freeze-dried mussels exposure, this one representing an in toto malrix, therefore areai food intake in human diet; - propose a TEF approach for PBDEs toxicity classification by integrating the overal! data produced by the project. Thus, the project will provide important results which will serve as a basis for improvement of the quality of life by appropriate regulatory and management actions_ D ORIGINALlTY OF THE PROPOSAL PROJECT Since the last decade, the occurrence of PBDEs was widely exploiled in several European and Asiatic countries and in USA, in Italy very little has been done on the topic and therefore only few infonnation have been collected 00 the environmentallevels, dielary exposure and toxicological effects related lo Ihese compouods. There is a lack of information on P8DEs contamination levels in Italian food, indeed in the Scientific Opinion on Polybrominated Diphenyl Ethers (PBDEs) in Food issued by EFSA in the fast August no data were submitted fram Ilaly_ Therefore, there is the need to improve the knowledge on PBDEs contamination paltems and levels in arder lo evaluate human and wildlife exposure and lo assess the related risk which could, at Ihe end, bring to the definition of residue limits to be fulfilled by the European Commissiono The use of mussels as sentinel organisms for the assessment of!he levels of persistent organic poflutants (POPs) and also of inorganic contaminanls in the environmenl has been frequently used worldwide (Mussel Wateh Projects). However, none of Ihese sludies has been conducted in Italy sioce now, especially on PBDEs, therefore valuable results are expected to come oul from this survey within the project. Moreover mussels, olher their use as iodicators enabling the measurement of cootaminants accumulation because of their sessile characteristics, are mainly a food and therefore Iheir examinatioo may provide important information 00 the possible human exposure to PBDEs as a consequence of consumption. Therefore sensitive and specific anatytical procedures are required to measure concenlrations al the sub ppb level. Gas chromatography is the elecled separation technique which, coupled with tandem mass spectrometry is bringing to the replacemenl of the more costly HRGC-HRMS by the HRGC-MS/MS methocls for trace anafysis of micropoflutants in various environmentat matrices, since il provides comparable selectivity and sensitivity. A more accessible and less expensive GC-MS/MS (triple quadrupole) method will be used as valuable analytical approach for the detennination of P8DEs in fish, rnussels and feed with!he aim to issue a validated Standardized Operating Procedure (SOP). The toxicological profile cf many PBDEs is 100 incomplete and insufficient to pericrm an adequate human and-ecological risk assessment. In this project the studies 00 zebrafish will be conducted improve the understanding of the toxic effects and poleotial health risks cf P8DEs, in parlicular by investigating the developmental malfonnalions and behavioural changes during the earty-life stages, as well as the histological modifications in gonads, liver and thyroid. The analysis following exposure to single PBDEs, namely BDE-47 and BDE-209, and to a PBDEs mixture ofthe B more relevanl congeners identified by EFSA, will also contribute to better investigate possible addilive/synergistic effects of Ihese compounds. As these PBDEs occur in Ihe environment in complex mixtures,!he identification of biological and toxicological structure-activity relalionships is of great importance for understanding the mixture laxicity cf this group of chemicals. In addilion, there is very liltle information available regarding useful biological or toxicological markers of low-ievel exposure to PBDEs and, in this context, the use of rodlet cells (RCs) and macrophage aggregates (MAs) may represenl a somehow innovative and interesting approach. Particular attention will be given to Ihe associaticn of these biomarkers with aoy signs of paraphysiological or pathological degeneration of liver, tyroid and gonads induced by chronic exposure to PBOEs. In particular, numeric and/or, morpho-densilometric paramelers related to MAs, such as mean, mode, minimum and maximum levels of grey, area, perimeter, circularity and fractal dimensions wifl be correlated lo time of PBDEs exposure. PBDEs mode of action, likewise dioxins and dioxin-like PCBs, has been mainly related to Iheir interaction with AhR with consequent induction of importanl liver enzymes such as Phase I cytochrome P450 mainly belonging lo 1A 1, 1A2 and 3A 1 families. Other mechanisrns at human liver level have been poorly invesligaled whereas effects 00 other organs of the digestive syslem have noi been coosidered at all, despite the importaoce of this system in foocl and food contaminants processing. Therefore, a comprehensive evalualion of possible effects at molecular level 00 Ihese orgaos is advisable and will be periormed within this praject by using!wo humao in vitra cellular models of liver and colon origino These celliines will be exposed either lo BDE-47 or BDE-209 as well as to a mixture of the 8-EFSA more rele.vant congeners at environmentally realistic concentrations as delermined in mussel samples. This aspecl is fundamental in order to mimic areai exposure scenario. In addition, both celllines will be exposed also to the freeze-dried mussels lo compare the effects of PBDEs as purified compounds and wilhin food using the in tolo matrix, where polential protective factors may exists. The analysis of the gene expression profiles in these samples wifl highlighl molecular larget stili noi investigated moreover helping to define possible biomarkers of early PBDEs exposure in two main larget organs. Results from this study will fili gaps in the knowledge af human exposure to PBDEs and in the fulure may be used in combination toxicological data to delermine health standards. E Methodology E1 Study Design with emerging The project will involve the analytical study of the PBDEs in mussels and then, once known the nalural conlamination levels, the in vivo (zebrafish) and in vitro (human celllines) studies will be periormed. The project will be divided into!wo Work Packages (WP), an Analytical WP and a Toxicological WP and seven phases (P). WP1: Analytical studies P1 (1-12 months): development and validalion of GC-MS (MS/MS) analytical melhods for!he analysis of the eight PBDE congeners considered by EFSA of primary interest (B-EFSA congeners: BDE-28, -47, -99, -100, -153, -154, -183 and -209)_ Elaboralion of the SOPs. P2 (6-18 months): harvesting of mussels samples from the Adriatic Sea cost and samples preparation.

15 /~~:."~;~~~~ P3 (12-18 months): analysis of mussel samples and assessment of lhe contamination levels i~:ç/\-'c::'.'''''~'};.~.',_.:~ ~-~\ P5 (18-32 months): quantificalion of single PBDEs and PBDEs mixlure in feed and in whole zebrafish tissues {treated and<untreated),,foltowing the.: ~ in vivo experimental steps. l~~~<,-. P7 (32-36 months): Elaboration of resulls and dissemination. \>i".j \-~:'-". WP2: Toxicological Studies '-. -'. 'A' ; P4 (1B-32 months): toxicological studies using zebrafish as in vivo model. Exposure of zebrafish to dietary relevant levels of sinfilè-bbdés (BDE-47 and BDE-209). PBDEs mixture (B-EFSA congeners) and freeze-dried mussels via diet. Evaluation of endocrine and neurobehavioral effects, histological modificalions and identification of cellular biomarkers. P6(1B-32 months): toxicological studies using two in vilro models: human hepalocellular carcinoma (HepG2) and human coloreclal adenocarcinoma (OLD-1) cejl/ines. Cells exposure to dietary relevant levels of single PBDEs (BDE-47 and BDE-209), a PBDEs mixture of the 8-EFSA congeners and freeze-dried mussels. Evaluation of the effects by gene expression profiling. P7 (32-36): Elaboration of results and dissemination. E2 Preliminary Data We don't have worked yet on PBDEs mussels contamination assessment or in vivo and in vitro PBDEs toxicological studies, however we have consolidated experience on PCBs which share with PBDEs some structural and toxicological characteristic. In the IZS Umbria e Marche was involved in a PCBs survey in mussels from the Marche (11]. Eighteen PCB congeners were measured lo estimate the total PCBs concenlrations and lhe contamination pattems. The predominant congener was always the H6CB-153 followed by H6CB-138. They, together, made up 35-40% of total PCBs in all sampling sites. The effects of three PCBs mixtu"res, one dioxin-like and two non-djoxin like PCBs, were evaluated on a human cellular model far fetal male external genitalia development [12). Treated cells were analysed for their gene expression profiles: the three mixtures modulated different patterns of genes moreover highlighting that the two mixtures of officialty c1assified non-dioxin like PC-Bs actual1y have different modes of aetion. The results suggesl that the official PCBs classification may be revised taking into account differenl modes of aclion within non-dioxin like PCBs E3 Bibliographic Data Relative scarce are the data available on PBDEs mussels contamination in Mediterranean Sea. Mussels (Mitylus edulis) from the French coasl showed that the three,prevalent congeners were BDEs 47, 99 and 100 [13]. M. edulis collected in Korea reported a similar pattem with BDE-47, the dominant congener, followed by -99 and 100 [14]. In M. edulis from Bo Sea (China), the BDE~209 reported the highest concentratlon [15]. No data in mussels from Italian coasts were found. Only few sludies explored PBDEs effects on development, reproduction or neurobehavior in zebrafish. Data on long+term chronie exposure at relatively low doses are lacking and the few information available are on the evaluation of toxic effects upon exposure to single PBDE congeners. HepG2 are largely used in the scientific community to study hepatic toxicological effect. However, regarding PBDEs, only the interactions with the AhR and the activation of eytochrome P450 enzymes have been investigated in this celliine [16]. Para ilei mechanisms, such as oxidative damage [17] or interaction wilh the Pregnane X receptor [18] have been poor1y studied, whereas other possible modes of action, such interactions with other nuclear receptors, have not been investigated at ali. No data are available on DLD-1 cells, an in vitro model from the colon. No gene expression profiling has been perfonned on both celilines following PBDEs treatments especiajly at environmentally relevant concentrations. [11] Piersanli, A Organohalogen Compound. 68: (12] Tail, S. et al, Reproductive Toxicology. 32:1-14. [13] Johansson, I Chemosphere, 64: [14] Ramu, K Chemosphere, 148: [15] Zhen Wang Chemosphere, 74: [16] An, J et al Environmenlal Toxicology, 26(6): (17J Fe<y, Y el al Toxìcology, 264(1-2):45-51 (18] Lundgren, M. et al, Toxicol Lett, 184(2): E4 Work Melhodology Analytical determinations Methods development Focus will be on the analysis of the mentioned 8-EFSA PBDEs congeners at sub-ppb levels. Samples wil1 be freeze-dried and then the conlaminants extracled using Pressurized Liquid Extractor (PLE)_ Clean.up methodologies like Selid Phase Extraction and Gel Permeation Chromatography will be applied. The analytical methods wlll be set up following the performance criteria reported in 1883J2006/EC for dioxin-like PCBs and dioxins as well as the principles Methods validation cf green chemistry. The analytlcal methods will be validated in mussel, fish tissue and feed to estimate lheir main performances characteristics: precision (repeatability and intralaboratory reproducibility), trueness, limit of detection (LOD), limit of quantification (LOQ), specificity, ruggedness and measurement uncertainty. The validation pian will involve repealed experiments at different spiking levels in the whole measurement field. Mussel Sampling The harvesting will be canied over twenty sampling points c1assified alon9 the Marche coast for the mussel survey in the frame of 853/2004/EC and 854/2004/EC. Each point will be sampled five times during each sampling campaign and two campaigns will take piace in a year: once in the prespawning and once in the post+spawning periodo Each sample will be made of specimens of the same dimensions (presumably same age) selected on the basis of the shell length. PBDEs analysis PBDE concentrations will be measured in the sampled mussels, in commerciai feed, in contaminated feed and in freeze-dried mussels used as source of exposure for toxicological trials. Zebrafish tissues will be also analysed before, during and after dietary exposure lo PBDEs. Ali the analysis will be performed in batehes with the appropriate quality controls. u-

16 In vivo toxicological Animals tests PBOEs exposure tests will be done using zebrafish (Oanio rerio), as recommended by the Organization for Economie Cooperation and Oevelopmenl (OECO) guidelines. Wild-!ype adult zebrafish will be initially purchased from a commerciai source and maintained in a laboratory environment far one month prior to breeding. Embryos and larvae witl be obtained by natural mating in spawning tanks and raised in optimal conditions. Experimental design Tests whllast 240 days post hatching (dph). From 4 dph to 20 dph larva! fish witl be fed with commerciai fish powder. The exposure will start from 20 dph and 1 rnonth after the exposition Ihe behavioural text wilt be done. The fish will be euthanized and analysed at fixed urne interval (lo (20dph), t 30 (50dph), t 70 (90dph), t 220 (240dph)). For Ihe PBOEs treatecl diets BOE 47, BOE 209 and the PBOEs rnixture wilt be added lo a commerciai formulaled fish food. Far each PBOEs standard Ihree trials will be prepared: one group fed with untreated feed (controj) a second fed with PBOEs contaminated feed (testing group) and a third group fed with feed contaminateci by the solvent used lo prepare the P8DEs solulions (solvent contrai). The concentrations of P8DEs in food witl be dose to median concentration measured in mussels in WP1. The last zebrafish group wilt be fed by freeze-dried mussels collected during the sampling campaign. Histological preparation and gametogenesis analysis Fish liver will be sampled for ali fish, according to experimental design, and processed for light microscopy and TEM. Quali-quantitative analysis of RCs and MAs witt be performed ali over the treatment period at fixed time interval (to, t3d, t70, 1220). Moreover, gonads and thyroid will be also scanned for pathology and assessed for their development. In vitro loxicological tests Celi cultures and lreatments The human hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (OLO.1) celliines will be grown in RPMI1640 medium wilhout phenoj recl, with 10% fetal bovine serum, 2mMI-glutamine, 100Ulml penicillin and 100g/ml streptomycin. Cells witr be maintained at 3rC and 5% C02. Freeze-dried mussels wi1l be reconstituted, filtered to obtain sterile solutions. Mussels and P8DEs standards will be properly diluted in culture medium. 80th celliines will be exposed to BOE-47, BDE-209, 8-P8DEs mixture, reconstituted freeze-dried mussels or medium alone as control. PBOEs concentrations used will be as those detected in Italian mussels. Treatments will be performed in triplicate, far 72h in arder to mimic a chronic exposure. Then, cetls will be trypsinized, collectecl, added of a Iysis buffer preventing RNA degradation and stored at -80 GC. RNA extraction and microarray analysis Total RNA will be exlracted from each sample. A gene expression profiling will be performed using Agilent Whole Human Genome microarrays featuring genes. Experiments will be performed following a dye-swap design. Control and Ireated samples wiu be co-hybridized on microarray slides and incubated at 65 GCfor 17h in a rotating incubator. Slides will be then read far their fluorescence in an Agilent scanner. Data will be analysed by bioinfonnatics tools to detennine the list of differentially expressed genes, as well as the pathways and networks indicating interrelations among groups of modulated genes. Real-time PCR Microarray results will be validaled by performing Real-time PCR on a subset of modulated genes. Reactions will be performed on RNA sample retrotranscribed to cona in presence of specific primers. Experiments will be performed in duplicate on a Stratagene MX5005P Ihermocycler inslrument. ResuJts will be analysed far their significance with the JMP 7.0 statisticaj software. ES Possible Criticai Aspects The first crilical poinl could be the harvesting in the winler due to the unfavourable almospheric conditions. In order lo avoid the lacking of single samples we will collect mussel in a Japse of 20 days. The second criticai poinl could be the mortality of zebrafish during Ihe first phase. For this raison, we have decided to start the exposure texts from 20 dph, when the larval survivorship is more stable. A criticai issue in in vitra toxicological experiments may be the filtration and the solubilisation lo a proper dilution, to be used in celi treatments, af freeze-dried mussels. The same procedure, as applied to infant foittlulasin the European Network of Excellence CASCAOE (155 as Partner), will be used. E6 Milestones Milestone (M) M1 (12th month): SOPs for the analysis of PBOE congeners in fish, feed and mussel tissues; beginning cf the harvesting campaign, analysis and preliminary resujts. M2 (18th month): ending of the harvesting campaign; analysis and results; In vivo and in vitro toxicological studies slart; midterm report of the research project. M3 (24th month): preliminary results of in vivo and in vitro toxicological studies and on PBOEs. M4 (32nd month): fina I results of in vivo and in vitro toxicologicaj studies; M5 (36nd month): research final report; diffusion of results by pubjication of papers and oral presentation at symposia. F TOOLS;INSTRUMENTS ANO COLLABORATION The Institution which will supervise and coordinale the activities of the project ISthe Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche (IZS-UM), an Italian governmental velerinary agency founded in 1936 and operating in the frame of the National Health System. The IZS-UM fs part of a network of ten similar institutions operating in other regions of the nalional tenitory. The ffelds of in!erest are manly the animai health and the food safety invojving the synergic collaboralion of differenl competences: veterinary, chemical, biological, biotechnological, epidemiological elc. The IZS-UM among many other structures and laboratories has its self labora!ories operating in the field of analytical chemistry applied to food anatysis and involved in the control of drugs and anabolic residues, persistent organic pollutants, pesticldes, micotoxins, biotoxins, inorganic conlaminanls in feed and food of animai origino The chemistry laboratories have quite advanced technical equipment both in the field of sample preparation and in the instrumental analytical delermination: Accelerateci Solvent Estractors (ASE), Gel Permeation Chromatography (GPC) syslems, Microwave ovens, automalic syslem far Solid Phase Extraction (SPE) cleanup and automatic dilutors. The analytical instruments available in the IZS-UM laboralories are: Gaschromatographic systems (GC) wilh seleetive detectors.like Electroncaplure Oetectors (ECO), Nitrogen Phosphorus Oetectors

17 /0~':~:~~ I~._:{;:;~~:.~..::.~,',',.. :'<'~>\ I "','") \ (NPD). Flame lonization Detectors (FIO), single quadrupole and ionie trap Mass Speetrometers (MS); Iiquid ehromaiograp~;~ystem;-;~'~~~) with '.~.;_'-, ;('IJ Diode Array (DAD) and Fluorescence Detectors and triple Quadrupole MSs. Far the determination of inorganic contaminant~~heizs-ùiv1.f[as an Inducled Ccupled Plasma MS (ICP~MS). The researchers working in the institution are ali well skilled in the topics of activity à~:p~sides Ihe',-...,.~.I \". ".:.. / institutional control of the compliance to lows of the food samples laken in lhe frame of offigial monitoring programs, the researèh:r:epre;ients oile'or ", ~- -,... the foremosl aims therefore the researchers have a150 experience in managing research projects. '. o.' The network of the Italian Isituti ZooprofilaHici Sperimentali grves the opportunity of an effective exchange of experience and expertise among the ten institutions. Moreover the scientific and experimenta! collaboralion involves many other organizations, among which universities, CNR, 155, research centres and reference laboratories. In the Ancona department of the IZS-UM has been recognised in 2003, by the Minislry of Health. lhe National Reference Laboratory for the Biological and Chemical Control of Bivalve Mussels and therefore a lot of work is done on the topic of chemical and bacterial analysis of mussels (Gazzella Ufficiale della Repubblica Italiana N. 81 del 07 Aprile 2003)_ The Ancona laboratories of lhe IZS-UM are also involved in lhe classification and in the syslematic survey of the areas where harvesting of mussels and clams far commerciai use is authorised. The project will involve the collaboration of researchers of the Teramo University, Veter;nary Medicine experts in toxicology and anatomical pathology and lhe researchers of the Istituto Superiore di Sanità (ISS) experts in food safety and endocrine disruptors risk assessment by in vitro toxicological studies. The Teramo group includes veterinary researchers with expertises in phannacotogy, toxicology, anatomical pathology and histology proven by numerous publications in indexed and impacted journals. The engagement into the project of researchers belonging to different scientific disciplines ensure a multidisciplinary approach. The contribulion of Teramo group will be focused in planning and conducting chronic toxicity trials on fish, in the study of toxicological pathology and in the identification of cellular biomarkers of effect. The Teramo group has its self taboratories operating in the field of b;ology, toxicology, anatomical pathology and veterinary medicine. The laboratories are well equipped to implement in vivo toxicological experimeols with ali the necessary technical facilities available. ISS is the main ins!itution of the National Health System as regards human health. Among the departments within ISS, the Dept. of Veterinary Public Health and Food Safety is especially involved in chemical and microbiological risk assessmenls in feed and food being also the National Focal Point of the European Food Safety Agency (EFSA). In particular, researchers in the Food and Veterinary Toxicology Unit focus their activities on the evaluation of potential adverse effects on human health following exposure to a number of compounds acting as endocrine disruptors. The laboratory is fully equipped to perform celi and molecular experiments and analysis. moreover possessing highly cost facilities for microarray and Real-time PCR assays. G RELEVANCE ANO IMPACT FOR THE NATIONAL HEALTH SYSTEM (SSN) The European Commission (EC), as already mentioned, asked the European Food Safety Authority (EFSA) to give a scientific opinion on PBDEs in food and in August 2011 the EFSA Panel on Contaminants in the Food Chain (CONTAM) published the Scientific Opioion 00 Polybrominaled Diphenyl Ethers (PBDEs) in Food [19]. Following the advice of the CONTAM Panel, a monitoring program was carried out starting trom 2006 and results obtained from the analysis of 19 PBDE congeners in 3971 fooosamples were provided to EFSA by eleven European countries, covering the period tram 2001 to No data were submitted from Italy because of a lack of information: the PBDEs contamination level in Italian food has been. to dale, poorly investigaled. Moreover, among the eight PBDE congeners (BDE-28, -47, -99, -100, -153, -154, -183 and -209) considered to be of primary interest for dietary exposure, relevant toxicity data were available oniy for BDE-47, -99, -153 and -209, therefore lhe risk assessment could be managed only for them. The EFSA Scientific Opinion strongly recommend to continue lhe surveitlance of PBDEs and to pertorm any further toxicological study on PBDEs taking into account the most relevant congeners to human exposure. The EC is currently eonsidering the establishment of maximum residue Iimits for these pollutants and is encouraging Member States to carry oul measurements to define the low background levels. The Tolerable Intake Level (Tolerabie Daily or Weekly Intake, TOlfTWl) has not been detennined for PBDEs by expert bodies such as the European Food Safety Aulhority, because there is yet insufficient information available on their toxicity and their occurrenee in food. It is, therefore, necessary to inerease the knowledge 00 PBDEs contamination levels in the Italian environment and in food matrices as well as to improve the toxicological assessment of these emerging contaminants. As a matter of fact without knowing PBDEs contamination levels it is not possible to make a risk assessment for the Italian populalion. The study of mussels as sentinel organisms can be of great help in the assessment of the aquatic environment. Overall, this project will g;ve a great contribution in the establishment of PBDEs contamination levels in primary senlinel organisms like mussels in Adriatic Sea. Moreover, relevant contribution lo the elucidation of PBDEs potential adverse effecls on human and wildlife will come trom both in vivo and vitro toxicological studies. A valuable tool to discriminale among PBDEs potential and relative toxicities would be the establishment of toxicological parameters to define accepted Toxicity Equivalency Factors (TEFs) for the different congeners. The TEF approach is of great importance in the risk assessment of chemical contaminants and is a very usefullool at regulatory level, but il is available only for some classes of substances (dioxin and dioxin-like compounds). This interdisciplinary study which will provide exposure, loxicological and mechanistic data may really serve as a basis for the TEFs definition. [19] Scientific Opinion of EFSA panel on Polybrominated Diphenyl Ethers (PBDEs) in Foed. H:COMPLIANCE Ves or No yes Reasons WITH THE CALL OBJECTVES The present project deals with developing new reliable analytical methods for PBDEs analysis, their measurement in mussels to study lhe accumulation capacity and the exploitation of their toxicological properties by in vivo and in vi!ra studies. I PROPOSAL BUDGET

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